The objective of this revision application is to extend the scope and capabilities of the parent grant by analyzing the protein-protein interactions occurring in the early steps of autophagosome formation using cutting-edge cell imaging techniques combined with TALEN technology. The molecular mechanisms underlying the formation of the early membrane structure of autophagosome called the isolation membrane remains mystery and it is a fundamental question in cell biology. The isolation membrane starts to emerge from the endoplasmic reticulum, and is expanded to form the double-membrane vesicular structure called autophagosome that encircles captured macromolecules. Recent studies from my group revealed that mTORC1, the ULK1-Atg13-FIP200-Atg101 complex, and the Beclin 1-PI3KC3 (hVps34)-Atg14L complex interact and form a large macromolecular interaction in a manner dependent upon nutrients and mTORC1 activity. It remains unclear what interactions are involved between the multi-protein complexes to regulate the isolation membrane formation. The well-appreciated question in the field must be greatly addressed by revolutionary cell imaging tools. Cellular imaging techniques continuously evolve and it is important to implement cutting-edge tools in the study. In the parent grant, we proposed three specific aims: (1) to determine how mTORC1 negatively regulates the ULK1-Atg13-FIP200-Atg101 complex; (2) to define the role of Atg13 in autophagy induction; (3) to determine how ULK1 regulates the Atg14L-containing PI3KC3 complex. In this revised application, we have added new cell imaging tools and genetic tools to strengthen each aim. We will use microscopic tools for high-resolution live cell imaging, FLIM/FRET and nanoscope techniques to investigate how the autophagy protein complexes are recruited in a hierarchical manner and how they colocalize in proximity during the autophagosome formation and how the interactions are regulated by phosphorylation of autophagy proteins. This revised study includes the TALEN technology to generate KO and KI cell lines. This powerful genetic tool will be essential to generate cellular systems to study post- translational modifications, such as phosphorylation, overcoming the concern with use of recombinant protein overexpression. As demonstrated in its powerful, efficient applications in many cellular systems, the TALEN technology will be very useful in autophagy field in dissecting the autophagy pathway. The new tools introduced in this revised application will resolve previously-unseen protein-protein interactions, especially dynamic interactions, and define the recruitment of autophagy proteins in a hierarchical manner, thus enabling us to better understand the processes and mechanisms of the formation of autophagosome.